Biosensor comprising a lipid membrane containing gated ion channels

ABSTRACT

A biosensor has a lipid membrane ( 7 ) containing gated ion channels sensitive to the presence or otherwise of an analyte molecule in a sample applied, in use, to a first side of the lipid membrane ( 7 ). The lipid membrane ( 7 ) is disposed between a pair of electrodes ( 1, 2 ) in which a first layer of porous gel ( 4 ) is applied to the first side of the lipid membrane ( 7 ).

This invention relates to a biosensor, and in particular to a biosensorof the type which operates by detecting or measuring the transport ofions across a lipid membrane.

Biosensors based on the use of a gated channel protein spanning abilayer membrane are of considerable interest. Each individual bindingevent can give rise to the passage of as many as 10⁹ individual ionsthrough the channel during a practical measurement interval. Also, theoperation of distinct channels is essentially independent and thecurrents through them combine linearly. These two factors inspire thehope of a general principle for sensing biomolecules which displays bothexcellent sensitivity and high dynamic range.

In order to achieve high dynamic range it is necessary to choose channelproteins which open in the presence of the target biomolecule. This isthe case for a number of naturally-occurring channel proteins, typicallyneurotransmitter receptors in nerve cells. In a more generallyapplicable approach, robust ungated channels, particularly thegramicidins, are bound chemically to antibody molecules in such a way asnormally to obstruct the channel, and to unblock it when an antigenbinds.

The electric charge transported through these channel proteins consistsphysically of solvated ions. In order to allow further processing, theions must be exchanged for the flow of electrons through a wire atelectrodes located at both the front and rear of the bilayer. In oneknown approach, the bilayer is located immediately adjacent to the rearnoble-metal electrode. It is not clear where the ions flow to. If theydischarge at the electrode, the associated chemical changes willinevitably lead to degradation.

In another known approach, there is electrolyte behind the bilayercontained in a gel. The bilayer is fabricated by a standard techniqueacross an aperture adjacent to the gel. The gel provides some physicalsupport for the bilayer, so that it is able to withstand quite vigorousagitation of the test liquid. However the bilayer cannot be dehydratedand must be formed immediately prior to the measurement in the aqueousmedium to be monitored. It is directly exposed to the medium and cannotwithstand contact with a solid.

There has now been devised a novel form of biosensor based onmeasurement of ion transport across a lipid membrane which overcomes orsubstantially mitigates the disadvantages of known forms of suchbiosensor.

According to a first aspect of the invention, a biosensor comprises alipid membrane containing gated ion channels sensitive to the presenceor otherwise of an analyte molecule in a sample applied, in use, to afirst side of said lipid membrane, the lipid membrane being disposedbetween a pair of electrodes, wherein a first layer of a porous gel isapplied to the first side of the lipid membrane.

The biosensor according to the invention is advantageous primarily inthat the first layer of porous gel applied to the lipid membraneprotects the membrane from dehydration and physical damage caused bymechanical contact, yet still permits molecules contained within thesample access to the lipid membrane. Because the membrane is notdestroyed by drying of the biosensor, the biosensor can be packaged andstored in the dry state, for rehydration immediately prior to use.

Preferably, a second layer of gel is also applied to the second side ofthe lipid membrane, to further protect the membrane and to provide thenecessary separation from the adjacent electrode and to accommodate areservoir of ions required by that electrode.

The gel is preferably a biocompatible and porous gel, most preferably ahydrogel. Suitable gel materials include agarose, dextran, carrageenan,alginic acid, starch, cellulose, or derivatives of these such as egcarboxymethyl derivatives, or a water-swellable organic polymer such aseg polyvinyl alcohol, polyacrylic acid, polyacrylamide or polyethyleneglycol. A particularly preferred gel material is agarose. Other gelmaterials considered particularly suitable include polyacrylaimide gels.

The thickness of particularly the first layer of gel is preferably suchas to permit diffusion of biomolecules of approximately 1 kD to occur inreasonably short time periods, eg less than 5 minutes, more preferablyless than 2 minutes. The first and second layers of gel preferably havethicknesses of less than 5 mm, eg 0.1 to 2 mm, most preferablyapproximately about 1 mm.

The lipid membrane is preferably a bilayer of amphiphilic molecules,most preferably one or more phospholipids, eg phosphatidylcholinesand/or phosphatidylethanolamines. The lipids may have hydrocarbon tailswith chain lengths of C₁₂-C₂₂, most preferably C₁₂-C₁₈. A particularlypreferred phospholipid is dioleylphosphatidylcholine. Other membraneforming molecules which may be employed include amphiphilic polymers, eghydrophobic polymer chains with hydrophilic side groups. One example ofsuch a polymer is a polysiloxane with phosphatidylcholine side groups.

Suitable molecules defining the gated ion channels are incorporated intothe lipid membrane, eg membrane-bound proteins.

Preferably a perforated sheet of an inert and impermeable material isinterposed between the lipid membrane and the second gel layer. Asuitable such material is polytetrafluoroethylene. The sheet ispreferably thin, eg less than about 100 μm in thickness, more preferablyabout 10 μm in thickness. The sheet is preferably formed with one ormore perforations of diameter 10-200 μm, more preferably about 50-100μm. The sheet of material permits the flow of current between theelectrodes only in the region of the perforation(s) in the sheet.

The lipid membrane may be formed by dissolving the membrane-forminglipid and the molecules defining the gated ion channels in a solvent andapplying the solution so formed to the second gel layer (or to theperforated sheet of inert material abutting the second gel layer). Anysuitable solvent may be used, provided that it is substantiallyimmiscible with water. Polar solvents, capable of initiating hydrogenbonds, are preferred since their use provides a strong driving force forcomplete coverage of the solution over the surface. A particularlypreferred solvent is chloroform. The solution preferably has aconcentration of 0.01 to 5% w/v, more preferably less than 1% w/v, egabout 0.2% w/v.

However, the method of forming the lipid membrane described above maynot always be suitable. For example, some ligand-gated channel proteinsmay be denatured by chloroform. One alternative method for the formationof the lipid membrane which may be suitable in such cases involvesforming an inverted micellar solution or emulsion containing themembrane forming lipid and molecules defining the gated ion channels,the micellar solution or emulsion having a hydrocarbon continuous phase.The hydrocarbon is preferably an alkane, most preferably hexane.

One functional ligand-gated channel protein for which the alternativemethod described in the immediately preceding paragraph may beapplicable is the nicotinic acetycholine receptor (nAChR—see G Puu etal, Biosens. Bioelectron. 10 (1995), 463). This neuroreceptor, with manyslight variations, is found in most animals with nervous systems, and avery rich source of supply is available in the electric organ of thecommon marbled ray Torpedo marmorata. A crude extract formed byhomogenizing the electric organ of the ray and centrifuging in a CsC1gradient to isolate the membrane-bound fraction has a continuous phasewhich is essentially aqueous. By reducing the proportion of water it ispossible to invert the emusion and to prepare from it an inverseemulsion with a hydrocarbon continuous phase having the characteristicsrequired for formation of the lipid membrane.

The electrodes are preferably noble metal electrodes of generallyconventional form. Most preferably the electrodes are silver/silverchloride electrodes formed on sheets of a suitable substrate such asmica. Preferably the electrode on the first side of the lipid membraneis formed with an aperture which serves as a sample introduction port.Where the device comprises a perforated sheet of material as describedabove, the aperture is preferably aligned with the perforation(s).

The invention further provides a method of qualitatively orquantitatively determining an analyte molecule in a sample, which methodcomprises applying the sample to the first layer of porous gel in abiosensor according to the first aspect of the invention.

The biosensor of the invention is most preferably assembled by coatingeach of two planar electrodes with a layer of porous gel, placing asolution or emulsion containing lipid molecules onto the second gellayer, and then placing one electrode on the top of the other such thatthe gel layers are diposed between them. Generally, the biosensor isassembled under the conditions such that the lipid membrane formsspontaneously. Monolayers of lipid will form at the interfaces betweenthe solution or emulsion and the respective gel layers. As the bulksolution or emulsion is expelled or evaporates from between the gellayers the two monolayers come together to form the lipid membranebilayer.

A preferred embodiment of the invention will now be described in greaterdetail, by way of example only, with reference to the accompanyingFigures, in which

FIG. 1 is a schematic sectional side view of a biosensor cell accordingto the invention;

FIG. 2 shows a bar histogram of cell resistances (logarithmic resistancescale) measured using a cell of the type shown in FIG. 1, with andwithout a lipid bilayer; and

FIG. 3 shows a scatter plot of measured cell resistance (log scale)measured using the cell of FIG. 1 after long exposure to gramicidin,versus the time taken for the resistance to reach its ultimate value.

Referring first to FIG. 1, a biosensor cell according to the inventionis formed between a pair of planar Ag/AgCl electrodes 1,2 formed on micasubstrates 1 a,2 a as described below. The upper mica substrate 1 a hasa 3 mm diameter aperture 3 through which test samples may be introduced.The space between the electrodes 1,2 is filled by first and secondlayers 4,5 of agarose gel (prepared as described below) separated by a10 μm thick PTFE sheet 6 on the upper surface of which is formed a lipidbilayer 7. The sheet 6 has at least one perforation 10. Silver wires 8,9are connected to the electrodes 1,2.

Formation of Gel Sheets

A mixture of 1% by weight agarose, 10% by weight glycerol, 0.1M NaCl,0.1M KCl and 0.01M CaCl₂ and the remainder ultrapure water were heatedto boiling point. While still liquid the mixture was pipetted into ahydrophilic glass mould or surface (typically 6 ml) and allowed to set.

The gel sheets 4,5 were 1 mm thick on initial formation. They wereallowed to dehydrate completely under laminar flow of ambient air(nominal 23° C. 50% relative humidity). After rehydration withelectrolyte (0.1M NaCl, 0.1M KCl, 0.01M CaCl₂ in ultrapure water) thegel was removed from its substrate.

Formation of Electrodes

The silver/silver chloride electrodes 1,2 were fabricated as follows.Freshly-cleaved pieces of mica 1 a,2 a were perforated as required(typically 3 mm diameter holes in a piece of a few centimetres in size).They were rinsed and sonicated separately with chloroform and methanol.A 12.5 μm-diameter silver wire 8,9 was placed on the surface which wasthen coated with silver dag and allowed to dry, securing the wire 8,9.The surface was electrolytically chlorided in 1M HCl at 9V using astainless steel counterelectrode, initially as the cathode for 10 s andthen as the anode for a further 10 s. The rehydrated gel layer 4,5described in the previous paragraph was placed in contact with thesilver side.

Assembly of Biosensor Cell

10 μm-thick PTFE film 6 was cut into cm-size pieces and perforated witha red-hot tungsten tip. The resulting holes were typically 50-100 μm indiameter.

A spreading solution was made up from L,α-dioleylphosphatidylcholine ata concentration of 20 g/l in chloroform.

A structure shown in FIG. 1 was assembled as follows. The perforatedpiece of PTFE 6 was placed on the lower electrode 2 with its gel coating5. 10 μl of spreading solution was spread over the PTFE 6 and a second,apertured electrode 1 with its gel coating 4 placed on top, ensuringthat the aperture 3 was aligned with a perforation in the PTFE sheet 6.

Measurements

For measurement, the electrolytic bilayer cells produced by the aboveprocedure were placed in a electrically-shielded box and a drop of testsolution placed over the mica aperture. The resistance of the cells wasmeasured with a Keithley Model 175 digital multimeter. To measure thevariation of current as a function of time, the cell current wasconverted to a voltage using a Bio-Logic BLM-120 bilayer membraneamplifier with a transimpedance of 1.0 GΩ. The output voltage wasdigitised by a Cambridge Electronic Design 1401 Plus multichannelanalyser and logged by a computer running the CDR program.

FIG. 2 shows the results of six measurements of the resistance of thecell prepared as described above, contrasted with measurements made inthe absence of a lipid membrane. As can be seen, the effect of the lipidmembrane is to increase the measured resistance considerably.

FIG. 3 shows measured resistance values for a cell prepared as describedabove, after long exposure to a 1 g/l solution of gramicidin-D. Theeffect of the protein is to reduce the cell resistance.

What is claimed is:
 1. A biosensor comprising a lipid membranecontaining gated ion channels sensitive to the presence or otherwise ofan analyte molecule in a sample applied, in use, to a first side of saidlipid membrane, the lipid membrane being disposed between a pair ofelectrodes, wherein the first side of the lipid membrane is coated witha first layer of porous get and the second side of the lipid membrane iscoated with a second layer of porous gel.
 2. A biosensor as claimed inclaim 1, wherein the gel is a hydrogel.
 3. A biosensor as claimed inclaim 2 wherein the first layer of gel is such as to permit diffusion ofbiomolecules of approximately 1 kD to occur in less than 5 minutes.
 4. Amethod of qualitatively or quantitatively determining an analytemolecule in a sample, which method comprises applying the sample to thefirst layer of porous gel in a biosensor according to claim
 3. 5. Abiosensor as claimed in claim 2 wherein the lipids of the lipid membranehave hydrocarbon tails with chain lengths Of C₁₂₋₂₂.
 6. A method ofqualitatively or quantitatively determining an analyte molecule in asarnple, which method comprises applying the sample to the first layerof porous gel in a biosensor according to claim
 2. 7. A biosensor asclaimed in claim 1 wherein the first layer of gel is such as to permitdiffusion of biomolecules of approximately 1 kD to occur in less than 5minutes.
 8. A biosensor as claimed in claim 7 wherein the lipids of thelipid membrane have hydrocarbon tails with chain lengths of C₁₂₋₂₂.
 9. Amethod of qualitatively or quantitatively determining an analytemolecule in a sample, which method comprises applying the sample to thefirst layer of porous gel in a biosensor according to claim
 7. 10. Abiosensor as claimed in claim 1 wherein the lipids of the lipid membranehave hydrocarbon tails with chain lengths of C₁₂₋₂₂.
 11. A method ofqualitatively or quantitatively determining an analyte molecule in asample, which method comprises applying the sample to the first layer ofporous gel in a biosensor according to claim
 1. 12. A method ofassembling a biosensor comprising coating each of two planar electrodeswith a layer of porous gel, placing lipid solution on to one of saidlayers and then placing one electrode on top of the other such that thegel layers are disposed between them and are separated by a lipidmembrane.
 13. A method as claimed in claim 12 wherein prior to theformation of the lipid membrane the membrane-forming lipid and themolecules defining gated ion channels are dissolved in a solvent.
 14. Amethod as claimed in claim 13 wherein the solvent is chloroform.
 15. Amethod as claimed in claim 12 wherein, prior to the formation of thelipid membrane the membrane-forming lipid and molecules defining gatedion channels are incorporated into an inverted emulsion with analkane-rich continuous phase.
 16. A method as claimed in claim 15wherein the alkane is hexane.